As is well-known, vitamin A is extremely important for the health of humans and animals. Among its essential functions are cellular differentiation and vision. Deficiency of Vitamin A can result in adverse effects on reproduction, growth, and the immune response. Vitamin A deficiency can also affect the eyes often resulting in blindness.
On the other hand, high levels of vitamin A can cause serious toxic manifestations; namely teratogenicity, chronic toxicity and acute hypervitaminosis. Some individuals may also show a genetic sensitivity to vitamin A, termed vitamin A intolerance, at intakes not much above those normally ingested. Thus, management of Vitamin A levels can have serious implications.
Presently in Third World nations, many young children are plagued by night blindness and other vitamin A-related diseases. It has been estimated that more than one million become permanently blind each year because of vitamin A deficiency.
Accordingly, for the above reasons, development of a fast and accurate method of measuring vitamin A levels is of international importance. This invention relates to a method of determining vitamin A levels in human blood serum which offers the advantage of smaller blood samples for analysis as well as less time and more simplicity.
Vitamin A (retinol) is normally transported in the blood as a complex with RBP (retinol-binding protein). RBP is a single polypeptide chain with a molecular mass of about 21,000 and has a single binding site for one molecule of retinol. The retinol-RBP further interacts strongly with another protein, plasma transthyretin (TTR or prealbumin) and normally circulates in plasma as a 1:1 complex (molar ratio) with TTR. These complex interactions and association of companion proteins with retinol have made determining retinol levels a difficult, time-consuming task.
The serum retinol concentration is the most commonly used indicator of vitamin A status. The preferred method for analysis of retinol is high-performance liquid chromatography (HPLC). The method involves collection of at least 200 .mu.l of whole blood, centrifugation within hours of collection, and keeping the samples in the cold during transport and storage. In the laboratory, the serum proteins are precipitated and retinol is extracted with organic solvents. The extracts are separated by HPLC and retinol is detected by UV absorbance or fluorescence. The limitations of this method are: (1) a large amount of serum sample (100 .mu.l or more) is needed, which is often difficult to obtain by capillary sampling and represents a large volume from infants, especially for neonates and low-birth-weight infants; (2) once separated from RBP, retinol is light-, oxygen- and heat-sensitive, increasing the likelihood of error during analysis; (3) processing time is relatively long.
Retinol is labile to light, oxygen, and heat, making it difficult to handle, especially when it is removed from the protection of its biological matrices. Extracted retinol decomposes rapidly even at subambient temperature when exposed to normal light.
In contrast, retinol is stable in frozen serum at -70.degree. C. for at least eight years. Retinol itself has a maximum absorbency at 325 nm and fluorescences at 425 nm. However, when retinol is bound to RBP the intensity of the fluorescence is enhanced ten to fourteen-fold and the fluorescence shifts to 465 nm. Additionally, these characteristics of retinol have provided encouragement for methods that would allow a direct determination of retinol in serum.
Current improvements along this vein include a micromethod involving gel-electrophoretic separation of serum, with subsequent estimation of the retinol-RBP complex by fluorimetric scanning of the gel. This method avoids solvent extraction, but is still limited by large sample requirements, long separation times and gel scanning, which make it of limited use for surveys of vitamin A status.
Recently, high performance size-exclusion liquid chromatography (SE-HPLC) with fluorescence detection has been used to measure retinol-RBP in animal and human serum. Compared to previous methods, less serum is required and preliminary sample treatment is avoided. However, a 0.05 ml blood sample is needed for analysis and 20-30 minutes of HPLC separation time is required. In addition, at least 1 ng of retinol-RBP is required for detection. These factors make it difficult to do minimicroassy, especially for babies and young children, where only small blood samples are available.
There is therefore a need in the art for a method to directly measure retinol in blood serum quickly, accurately, and without need for large blood samples.
The primary object of the present invention is, therefore, to develop a method to directly evaluate levels of vitamin A in human blood serum.
It is a further object of the present invention to provide a method to determine vitamin A levels in human blood serum which is accurate, fast, and easy.
It is a still further object of the present invention to develop a method to determine blood serum levels of vitamin A which requires smaller blood samples by use of Capillary Zone Electrophoresis than conventional methods.